Deorphanization of Malonyl CoA:ACP Transacylase Drug Target in Plasmodium falciparum (PfFabD) Using Bacterial Antagonists: A Piggyback' Approach for Antimalarial Drug Discovery

Quest for new drug targets in Plasmodium sp. has underscored malonyl CoA:ACP transacylase (PfFabD) of fatty acid biosynthetic pathway in apicoplast. In this study, a piggyback approach was employed for the receptor deorphanization using inhibitors of bacterial FabD enzymes. Due to the lack of crystal structure, theoretical model was constructed using the structural details of homologous enzymes. Sequence and structure analysis has localized the presence of two conserved pentapeptide motifs: GQGXG and GXSXG and five key invariant residues viz., Gln109, Ser193, Arg218, His305 and Gln354 characteristic of FabD enzyme. Active site mapping of PfFabD using substrate molecules has disclosed the spatial arrangement of key residues in the cavity. As structurally similar molecules exhibit similar biological activities, signature pharmacophore fingerprints of FabD antagonists were generated using 0D-3D descriptors for molecular similarity-based cluster analysis and to correlate with their binding profiles. It was observed that antagonists showing good geometrical fitness score were grouped in cluster-1, whereas those exhibiting high binding affinities in cluster-2. This study proves important to shed light on the active site environment to reveal the hotspot for binding with higher affinity and to narrow down the virtual screening process by searching for close neighbors of the active compounds.

Sequence-Structure Similarity: Do Sequentially Identical Peptide Fragments have Similar Three-Dimensional Structures?

The rapidly growing structure databases enhance the probability of finding identical sequences sharing structural similarity. Structure prediction methods are being used extensively to abridge the gap between known protein sequences and the solved structures which is essential to understand its specific biochemical and cellular functions. In this work, we plan to study the ambiguity between sequence-structure relationships and examine if sequentially identical peptide fragments adopt similar three-dimensional structures. Fragments of varying lengths (five to ten residues) were used to observe the behavior of sequence and its three-dimensional structures. The STAMP program was used to superpose the three-dimensional structures and the two parameters (Sequence Structure Similarity Score (Sc) and Root Mean Square Deviation value) were employed to classify them into three categories: similar, intermediate and dissimilar structures. Furthermore, the same approach was carried out on all the three-dimensional protein structures solved in the two organisms, Mycobacterium tuberculosis and Plasmodium falciparum to validate our results.

Insights into the Fold Organization of TIM Barrel from Interaction Energy Based Structure Networks

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or ``sequence conservation'' as the basis for their understanding. Recently ``interaction energy'' based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the ``interaction conservation'' viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.

Inter-domain interactions influence the stability and catalytic activity of the bi-domain protein tyrosine phosphatase PTP99A

The two protein tyrosine phosphatase (PTP) domains in bi-domain PTPs share high sequence and structural similarity. However, only one of the two PIP domains is catalytically active. Here we describe biochemical studies on the two tandem PTP domains of the bi-domain PTP, PTP99A. Phosphatase activity, monitored using small molecule as well as peptide substrates, revealed that the inactive (D2) domain activates the catalytic (D1) domain. Thermodynamic measurements suggest that the inactive D2 domain stabilizes the bi-domain (D1-D2) protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by few interactions at the inter-domain interface. In particular, mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent location to the so-called allosteric site of the canonical single domain PIP, PTP1B. These observations suggest functional optimization in bi-domain PTPs whereby the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme. (C) 2012 Elsevier B.V. All rights reserved.

PocketAnnotate: towards site-based function annotation

A computational pipeline PocketAnnotate for functional annotation of proteins at the level of binding sites has been proposed in this study. The pipeline integrates three in-house algorithms for site-based function annotation: PocketDepth, for prediction of binding sites in protein structures; PocketMatch, for rapid comparison of binding sites and PocketAlign, to obtain detailed alignment between pair of binding sites. A novel scheme has been developed to rapidly generate a database of non-redundant binding sites. For a given input protein structure, putative ligand-binding sites are identified, matched in real time against the database and the query substructure aligned with the promising hits, to obtain a set of possible ligands that the given protein could bind to. The input can be either whole protein structures or merely the substructures corresponding to possible binding sites. Structure-based function annotation at the level of binding sites thus achieved could prove very useful for cases where no obvious functional inference can be obtained based purely on sequence or fold-level analyses. An attempt has also been made to analyse proteins of no known function from Protein Data Bank. PocketAnnotate would be a valuable tool for the scientific community and contribute towards structure-based functional inference. The web server can be freely accessed at http://proline.biochem.iisc.ernet.in/pocketannotate/.

Positive Relationships between Association Strength and Phenotypic Similarity Characterize the Assembly of Mixed-Species Bird Flocks Worldwide

Competition theory predicts that local communities should consist of species that are more dissimilar than expected by chance. We find a strikingly different pattern in a multicontinent data set (55 presence-absence matrices from 24 locations) on the composition of mixed-species bird flocks, which are important sub-units of local bird communities the world over. By using null models and randomization tests followed by meta-analysis, we find the association strengths of species in flocks to be strongly related to similarity in body size and foraging behavior and higher for congeneric compared with noncongeneric species pairs. Given the local spatial scales of our individual analyses, differences in the habitat preferences of species are unlikely to have caused these association patterns; the patterns observed are most likely the outcome of species interactions. Extending group-living and social-information-use theory to a heterospecific context, we discuss potential behavioral mechanisms that lead to positive interactions among similar species in flocks, as well as ways in which competition costs are reduced. Our findings highlight the need to consider positive interactions along with competition when seeking to explain community assembly.

Similarity relations in visual search predict rapid visual categorization

How do we perform rapid visual categorization?It is widely thought that categorization involves evaluating the similarity of an object to other category items, but the underlying features and similarity relations remain unknown. Here, we hypothesized that categorization performance is based on perceived similarity relations between items within and outside the category. To this end, we measured the categorization performance of human subjects on three diverse visual categories (animals, vehicles, and tools) and across three hierarchical levels (superordinate, basic, and subordinate levels among animals). For the same subjects, we measured their perceived pair-wise similarities between objects using a visual search task. Regardless of category and hierarchical level, we found that the time taken to categorize an object could be predicted using its similarity to members within and outside its category. We were able to account for several classic categorization phenomena, such as (a) the longer times required to reject category membership; (b) the longer times to categorize atypical objects; and (c) differences in performance across tasks and across hierarchical levels. These categorization times were also accounted for by a model that extracts coarse structure from an image. The striking agreement observed between categorization and visual search suggests that these two disparate tasks depend on a shared coarse object representation.

Synthesis of a mixed-metal spinel, NiMn2O4, through site-selective substitution in MOF, (NiMn2){C6H3(COO)(3)}(2)]: synthesis, structure and magnetic studies

A mixed-metal metal-organic framework (MOF) compound NiMn2{C6H3(COO)(3)}(2)], I, is prepared hydrothermally by replacing one of the octahedral Mn2+ ions in Mn-3{C6H3(COO)(3)}(2)] by Ni2+ ions. Magnetic studies on I suggest antiferromagnetic interactions with weak canted antiferromagnetism below 8 K. On heating in flowing air I transforms to NiMn2O4 spinel at low temperature (T < 400 degrees C). The thermal decomposition of I at different temperatures results in NiMn2O4 with particle sizes in the nano regime. The nanoparticle nature of NiMn2O4 was confirmed using PXRD and TEM studies. Magnetic studies on the nanoparticles of NiMn2O4 indicate ferrimagnetism. The transition temperature of NiMn2O4 nanoparticles exhibits a direct correlation with the particle size. This study highlights the usefulness of MOF compound as a single-source precursor for the preparation of important ceramic oxides with better control on the stoichiometry and particle size.

Combination of similarity measures for time series classification using genetic algorithms

Time series classification deals with the problem of classification of data that is multivariate in nature. This means that one or more of the attributes is in the form of a sequence. The notion of similarity or distance, used in time series data, is significant and affects the accuracy, time, and space complexity of the classification algorithm. There exist numerous similarity measures for time series data, but each of them has its own disadvantages. Instead of relying upon a single similarity measure, our aim is to find the near optimal solution to the classification problem by combining different similarity measures. In this work, we use genetic algorithms to combine the similarity measures so as to get the best performance. The weightage given to different similarity measures evolves over a number of generations so as to get the best combination. We test our approach on a number of benchmark time series datasets and present promising results.

Low-pass filters and differential tympanal tuning in a paleotropical bushcricket with an unusually low frequency call

Low-frequency sounds are advantageous for long-range acoustic signal transmission, but for small animals they constitute a challenge for signal detection and localization. The efficient detection of sound in insects is enhanced by mechanical resonance either in the tracheal or tympanal system before subsequent neuronal amplification. Making small structures resonant at low sound frequencies poses challenges for insects and has not been adequately studied. Similarly, detecting the direction of long-wavelength sound using interaural signal amplitude and/or phase differences is difficult for small animals. Pseudophylline bushcrickets predominantly call at high, often ultrasonic frequencies, but a few paleotropical species use lower frequencies. We investigated the mechanical frequency tuning of the tympana of one such species, Onomarchus uninotatus, a large bushcricket that produces a narrow bandwidth call at an unusually low carrier frequency of 3.2. kHz. Onomarchus uninotatus, like most bushcrickets, has two large tympanal membranes on each fore-tibia. We found that both these membranes vibrate like hinged flaps anchored at the dorsal wall and do not show higher modes of vibration in the frequency range investigated (1.5-20. kHz). The anterior tympanal membrane acts as a low-pass filter, attenuating sounds at frequencies above 3.5. kHz, in contrast to the high-pass filter characteristic of other bushcricket tympana. Responses to higher frequencies are partitioned to the posterior tympanal membrane, which shows maximal sensitivity at several broad frequency ranges, peaking at 3.1, 7.4 and 14.4. kHz. This partitioning between the two tympanal membranes constitutes an unusual feature of peripheral auditory processing in insects. The complex tracheal shape of O. uninotatus also deviates from the known tube or horn shapes associated with simple band-pass or high-pass amplification of tracheal input to the tympana. Interestingly, while the anterior tympanal membrane shows directional sensitivity at conspecific call frequencies, the posterior tympanal membrane is not directional at conspecific frequencies and instead shows directionality at higher frequencies.

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

Isolation of a High Affinity Neutralizing Monoclonal Antibody against 2009 Pandemic H1N1 Virus That Binds at the `Sa' Antigenic Site

Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs) have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K-D = 2.1 +/- 0.4 pM) murine MAb, MA2077 that binds specifically to the hemagglutinin (HA) surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC50 = 0.08 mu g/ml). MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 mu g/ml) against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic) on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein `Sa' and `Sb' sites were independently mutated, localized the binding site of MA2077 within the `Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

Seismic site classification and correlation between standard penetration test N value and shear wave velocity for Lucknow city in Indo-Gangetic basin

Subsurface lithology and seismic site classification of Lucknow urban center located in the central part of the Indo-Gangetic Basin (IGB) are presented based on detailed shallow subsurface investigations and borehole analysis. These are done by carrying out 47 seismic surface wave tests using multichannel analysis of surface waves (MASW) and 23 boreholes drilled up to 30 m with standard penetration test (SPT) N values. Subsurface lithology profiles drawn from the drilled boreholes show low- to medium-compressibility clay and silty to poorly graded sand available till depth of 30 m. In addition, deeper boreholes (depth >150 m) were collected from the Lucknow Jal Nigam (Water Corporation), Government of Uttar Pradesh to understand deeper subsoil stratification. Deeper boreholes in this paper refer to those with depth over 150 m. These reports show the presence of clay mix with sand and Kankar at some locations till a depth of 150 m, followed by layers of sand, clay, and Kankar up to 400 m. Based on the available details, shallow and deeper cross-sections through Lucknow are presented. Shear wave velocity (SWV) and N-SPT values were measured for the study area using MASW and SPT testing. Measured SWV and N-SPT values for the same locations were found to be comparable. These values were used to estimate 30 m average values of N-SPT (N-30) and SWV (V-s(30)) for seismic site classification of the study area as per the National Earthquake Hazards Reduction Program (NEHRP) soil classification system. Based on the NEHRP classification, the entire study area is classified into site class C and D based on V-s(30) and site class D and E based on N-30. The issue of larger amplification during future seismic events is highlighted for a major part of the study area which comes under site class D and E. Also, the mismatch of site classes based on N-30 and V-s(30) raises the question of the suitability of the NEHRP classification system for the study region. Further, 17 sets of SPT and SWV data are used to develop a correlation between N-SPT and SWV. This represents a first attempt of seismic site classification and correlation between N-SPT and SWV in the Indo-Gangetic Basin.

Influence of rock depth on seismic site classification for shallow bedrock regions

Seismic site classifications are used to represent site effects for estimating hazard parameters (response spectral ordinates) at the soil surface. Seismic site classifications have generally been carried out using average shear wave velocity and/or standard penetration test n-values of top 30-m soil layers, according to the recommendations of the National Earthquake Hazards Reduction Program (NEHRP) or the International Building Code (IBC). The site classification system in the NEHRP and the IBC is based on the studies carried out in the United States where soil layers extend up to several hundred meters before reaching any distinct soil-bedrock interface and may not be directly applicable to other regions, especially in regions having shallow geological deposits. This paper investigates the influence of rock depth on site classes based on the recommendations of the NEHRP and the IBC. For this study, soil sites having a wide range of average shear wave velocities (or standard penetration test n-values) have been collected from different parts of Australia, China, and India. Shear wave velocities of rock layers underneath soil layers have also been collected at depths from a few meters to 180 m. It is shown that a site classification system based on the top 30-m soil layers often represents stiffer site classes for soil sites having shallow rock depths (rock depths less than 25 m from the soil surface). A new site classification system based on average soil thickness up to engineering bedrock has been proposed herein, which is considered more representative for soil sites in shallow bedrock regions. It has been observed that response spectral ordinates, amplification factors, and site periods estimated using one-dimensional shear wave analysis considering the depth of engineering bedrock are different from those obtained considering top 30-m soil layers.

Identification of source of heavy metal contamination in a site - a case study

Accidental spills and improper disposal of industrial effluent/sludge containing heavy metals onto the open land or into subsurface result in soil and water contamination. Detailed investigations are carried out to identify the source of contamination of heavy metals in an industrial suburb near Bangalore in India. Detailed investigation of ground water and subsurface soil analysis for various heavy metals has been carried out. Ground water samples were collected in the entire area through the cluster of borewells. Subsurface soil samples were collected from near borewells which were found to contain heavy metals. Water samples and soils samples (after acid digestion) were analysed as per APHO-standard method of analysis. While the results of Zn, Ni and Cd showed that they are within allowable limits in the soil, the ground water and soils in the site have concentration of Cr+6 far exceeding the allowable limits (up to 832 mg/kg). Considering the topography of the area, ground water movement and results of chromium concentration in the borewells and subsurface it was possible to identify the origin, zone of contamination and the migration path of Cr+6. The results indicated that the predominant mechanism of migration of Cr+6 is by diffusion.